This invention relates to a process for the continuous enzymatic treatment of lipid-containing compositions in a plurality of fixed bed reactors, and to an apparatus for practicing the process. More particularly, this invention relates to a process and apparatus for the continuous enzymatic treatment of lipid-containing compositions using a plurality of fixed bed reactors, wherein the flow of the lipid-containing composition remains substantially constant even as the enzymatic activity of a fixed bed changes over time, and even when a fixed bed is taken off-line such as for repair, replacement, or replenishment. Additionally, this invention relates to a process and apparatus that provides an unexpectedly significant increase in enzymatic activity by pretreating the lipid before it encounters the enzyme and operating the apparatus in a continuous process.
Fats are made of fatty acids attached to a three-carbon glycerol backbone. Fatty acids are made up of chains of carbon atoms with a terminal hydroxyl group. The hydroxyl groups can attach to one, two, or three of the hydroxyl groups on the glycerol backbone to form mono-, di-, or tri-acylglycerols, or fats. The functional and nutritional qualities of the fats will depend on a variety of factors including whether they are monoacylglycerol (MAG), a diacylglycerol (DAG) or a tri-acylglycerol (TAG); the number of carbons in the fatty acid chains; whether the chains are saturated, mono-unsaturated, or poly-unsaturated; whether any unsaturated double bonds in the chains are in the form of the cis or trans isomer; the location of any double bonds along the chains; and the positions of the different types of fatty acids relative to the three carbons of the glycerol backbone.
Lipids are a classification of a broad variety of chemical substances characterized as fats, oils, waxes, and phospholipids. Included within this broad classification are triglycerides, diglycerides, monoglycerides, fatty acids, fatty alcohols, soaps and detergents, terpenes, steroids, and vitamins A, E, D2, and K1. Lipids can be obtained from oilseeds such as soybeans, canola, rapeseed, sunflower, palm, and olives; animal products such as fish, pork, and beef; and synthetic compounds or synthetically derived compositions such as structured lipids for nutritional applications, oleochemicals for industrial and pharmaceutical applications, and biodiesel for energy. Vegetable oils are obtained by pressing or solvent extraction of the oil from the oilseed. The crude oils contain many minor components. Some of these components are detrimental to the performance or aesthetic properties of the oils; others, such as sterols and tocopherols, are nutritionally beneficial.
Lipids obtained from oilseeds (soybean, canola, etc.) by either solvent extraction or mechanical pressing can be refined to remove impurities that may contribute to undesirable colors and/or flavors in the finished product. Traditional refining includes treatment of the oil with sodium hydroxide to neutralize the free fatty acids, and removal of the phospholipids via centrifugation. The oil is then washed with hot softened water and centrifuged to remove the remaining soaps and phospholipids present in the oil. The “once refined” oil is then bleached with “bleaching earth” and filtered to adsorb the chlorophyll and chlorophyll derivatives as well as any remaining soaps, phospholipids, and trace metals present in the oil. The use of bleaching earths or clays for the removal of impurities in lipids is well known in the art. The first common name for the material was “Fuller's earth”. Present day bleaching earths may be neutral or acid activated. Mineral clays typically utilized are bentonite, montmorillonite, attapulgite, smectite, and/or hormite.
An alternative process which eliminates the water washing step entirely and replaces it with a treatment of silica gel to adsorb the residual soaps, phospholipids, and trace metals is well known in the art as “Modified Caustic Refining”. Pryor et al. U.S. Pat. No. 5,336,794 and Welsh et al U.S. Pat. No. 5,231,201 disclose a two-phase process wherein oil is first contacted with amorphous silica adsorbents to remove all or substantially all soaps or gums or both from the oil and reduce its phospholipid content, and then filtered through a packed bed of a pigment removal agent to decolorize the oil. A silica gel, 0.01 to 1.0 percent, is added to the oil in a slurry after the caustic treated oil is centrifuged. Silica gel products known to be useful for this purpose include those sold under the trademark TriSyl® (silica gel) by W.R. Grace & Co. as amorphous silica free flowing powders containing about 60 to 65 percent moisture with a particle size average of about 18.0 microns minimum, average pore diameter between about 60 and 5000 angstroms, and bulk density of about 500 kg/m3. The oil is mixed with the silica and then dried in a vacuum spray drier; the silica is then filtered out of the oil. If bleaching clay is already on the filter, the process is well known in the art as “Packed Bed Bleaching”. The moisture maintains the integrity of the silica pores and allows the impurities to be adsorbed inside the pore.
In recent years there has been increased interest in providing alternatives to the high trans fats and shortening products used in traditional food preparation. Traditionally, liquid oils were manufactured into functional fats containing solids for various margarine and shortening products by nickel hydrogenation. Such hydrogenation processes led to the formation of trans fatty acids. It is believed that fats having reduced trans fatty acids may provide certain health benefits to the consumer. Accordingly, many large food producers are replacing high trans fats with low or even zero trans fats compositions. Originally, efforts at providing low trans fats products focused on reducing the level of hydrogenation of the fat products. More recently, efforts have focused on changing the structure of a liquid oil to change the melting properties and functionality without changing the fatty acid composition or generating trans fatty acids. One method of achieving this is a process known as interesterification.
Interesterification is a known reaction of triacylglycerol structures whereby individual fatty acid structures at positions of the triglyceride being interesterified are interchanged on the glycerol moiety. This is at times referred to or recognized as a randomization wherein fatty acid moieties from one glycerol component of a triacylglycerol are exchanged with those of a glycerol component of another triacylglycerol. This results in triacylglycerol structures which have interchanged fatty acid moieties that vary from glycerol structure to glycerol structure. Art in this area includes Pellosa et al. U.S. Pat. No. 5,434,278, Doucet U.S. Pat. No. 5,908,655, Cherwin et al. U.S. Pat. No. 6,124,486, and Liu et al. U.S. Pat. No. 6,238,926.
The art of interesterification has developed to enable the production of, for example, triglyceride compositions which provide certain melt profiles that can be of interest in certain applications. Generally these are recognized herein as “structured lipids” to distinguish the interesterified products from physical blends of the same components that have not been subjected to interesterification. Swem, Bailey's Industrial Oil and Fat Products, 3rd edition, pages 941-970 (1964) described the reesterification of fatty acids and glycerol, mono- and poly-hydroxy alcohols, interesterification (acidolysis and alcoholysis), and transesterification of lipids via chemical methods.
Interesterification can be accomplished either chemically or enzymatically. Chemical interesterification is generally accomplished with a chemical catalyst such as sodium methoxide. While chemical interesterification can be less costly in terms of the catalyst, it has several distinct disadvantages. The sodium methoxide catalyst can be dangerous and difficult to handle. The resulting interesterification is random, and does not afford the manufacturer the degree of control that is preferred over the structure of the resulting product. Chemical interesterification also can result in relatively high oil losses. Art in this area includes Kaita et al. U.S. Pat. Application No. 2002/0010359, Bayense et al U.S. Pat. No. 6,072,064, Cooper et al. U.S. Pat. No. 5,399,728, and Stipp et al. U.S. Pat. No. 5,142,072.
In enzymatic interesterification, the enzyme catalyst is more costly than sodium methoxide, and it has low activity and low stability. But enzyme catalysts can afford a great deal of control over the structure of the final interesterified product. In particular the use of certain enzymes can result in interesterification specifically at the 1- and 3-positions along the glycerol backbone chain, exactly where it is most desired. While enzymatic catalysts were originally used only for high value-added products, they are now being used increasingly in the manufacture of commodity fats and fat blends.
Enzymes are complex proteins that produce a specific chemical reaction in other substances without themselves being changed, i.e., a biological catalyst. These biological catalysts are expressed or produced from various microorganisms. Enzymes suitable for use in the present invention include esterase; acylase; those enzymes that facilitate acidolysis reactions, transesterification reactions, ester synthesis, or ester interchange reactions; enzymes having phospholipase or protease activity, including thermostable and thermotolerant hydrolase activity; and polynucleotides. Microorganisms included within the art are Rhizopus, Aspergillus, Mucor, Geotrichum, Pseudomonas, Penicillium, Chromobacterium, Candida, Achromobacter, Alcaligenes, Corynebacterium, Humicora, Humicolo, Staphylococcus, Rhizomucor, Torulopsis, and Bacillus. Such enzymes produced from the above microorganisms are disclosed by Sugiura et al. U.S. Pat. Application No. 2001/0004462, Bosley et al. U.S. Pat. No. 5,773,266, Quinlan U.S. Pat. No. 5,658,768, Miyamoto et al. U.S. Pat. No. 5,461,170, and Myojo et al. U.S. Pat. No. 5,219,733.
In U.S. Pat. No. 5,508,182, Schneider et al. disclose numerous methods for producing amphiphilic compounds through the biocatalyzed reaction of a hydrophilic substrate, adsorbed onto a solid support, with a second substrate, which may be hydrophobic. Schneider et al. describes methods for producing isomerically pure 1,3-diglycerides and 1-monoglycerides, sugar esters, amino acid esters, peptides, and glycolipids, as well as phosphates of alcohols, carbohydrates, and nucleosides. The patent describes the adsorption of different substrates onto a solid support with an amino-protected amino acid or a carboxyl-protected peptide. Essentially, no reaction occurs without the presence of the substrate adsorbed on the support, examples 1 and 12, thus the support acts as the catalyst for the reactions. All of the examples given were batch reactions, including example 19 where the vinyllaurate (dissolved in t-BuOMe) is circulated through a packed bed column containing the adsorbed glycerol on the silica gel and the enzyme. The 1,3-dilaurate product is removed from the column by extracting with fresh t-BuOMe. It was not taught or suggested that the glycerol may be re-adsorbed and the reaction operated as a fixed bed reactor independent of the enzyme and/or silica gel. The amount of silica gel utilized in the disclosure ranged from 60 to 1000 percent of the substrate.
Enzymes utilized in the disclosure by Schneider et al. were from Mucor mihei, Pseudomonas fluorescens, Rhizopus delemar, Candida cylindracea, and Penicillium cyclopium. 
One particularly preferred enzyme catalyst is the lipase from Thermomyces lanuginosus. This enzyme is specific for the 1 and 3 sites on the glycerol backbone, and it is heat stable up to about 75° C. This enzyme, however, can be readily inactivated by radicals such as peroxides, certain polar impurities such as phosphatides and soaps, secondary oxidation products such as ketones and aldehydes, and trace metals. Thus, the quality of the oil feedstock is important. U.S. Patent Publication No. 2003/0054509 discloses the pretreatment of an oil prior to enzymatic interesterification with a silica. The amount of silica utilized in the examples was 172 percent of the enzyme utilized for the reaction (38 g of silica per 22 g of enzymes)
An immobilized granulated form of the lipase from Thermomyces lanuginosus is sold by Novozymes Corporation under the registered trademark LIPOZYME®TL IM. The product literature that comes with this enzyme product discloses a process of use comprising cooling the lipids to 70° C., pumping the lipids to a single reactor column or tank, and passing the oil through the column or mixing the oil with the enzyme in the tank. The lipids contact the enzyme in the column or tank and are continuously interesterified. The interesterified lipids may then be blended with other lipids, or deodorized, or shipped to the final customer.
Factors to be considered in designing an enzymatic interesterification process include whether it should be batch or continuous, whether it will include a single or multiple fixed bed reactor, if multiple fixed beds, whether the beds will be in series or in parallel, whether the flow rate will be variable or constant, how to control the extent of enzymatic conversions, and problems with potential cross-contamination. See e.g., “Chemical vs. Enzymatic Interesterification, by Wim De Greyt of the DeSmet Group, Belgium, presented at the IUPAC-AOCS Workshop on Fats, Oils and Oilseeds Analyses and Production, Dec. 6-8, 2004, available at http://www.aocs.org/archives/analysis/pdfs/degreyt-interesterification-modifieddgw.pdf. As disclosed therein, if a single fixed bed reactor is used, the enzymatic activity will decrease over time. The flow rate must be decreased in order to ensure that the reaction is allowed to go to completion. This requires a variable speed control pump, as well as regular monitoring of the conversion, and results in a low production rate at the end of the enzyme's lifetime. The process cannot be operated continuously because of the frequent need to remove and replace enzymes in the column. Often a catalyst bed must be replaced even if some of the catalyst in the bed is still active, resulting in waste of active catalyst. The size of the enzyme bed column is limited, because if the height is too great, the enzyme granules at the bottom may be crushed under the pressure exerted by the system pump, and if the diameter is too great, the granular material may distribute so as to form channels through which oil may pass without contacting and thereby reacting with the enzyme.
In a multiple fixed bed series reactor system, each fixed bed will have a different enzyme activity, with the first reactor bed having the lowest enzyme activity, and the last reactor having the highest enzyme activity. This is because the first reactor in the series absorbs more of the impurities and harmful components, thereby protecting the more active enzyme in the further reactors. Owen et al. disclose in U.S. Pat. No. 4,789,528 the operation of a sequential rotation of reactors in a multi-reactor fixed bed system utilizing zeolites in a petrochemical application to produce a variety of refined petrochemical products.
U.S. Pat. Publication No. US 2005/0014237 discloses a method of enzymatic interesterification wherein the feedstock is deodorized prior to contact with an enzyme, for the purpose of prolonging the half-life of the enzyme. Deodorization is described therein as typically the last step in the conventional oil refining process, and as being principally a steam distillation, during which substances with greater volatility are removed by high temperature under vacuum. Various substances removed by deodorization include free fatty acids and various flavor and odor compounds either present originally or generated by oxidation of fats and oils. Also removed are the substances formed by the heat decomposition of peroxides and pigments.
As reported by Ten Brink et al. in US 2005/0019316, JP 08000275 discloses that a pre-treatment of 2 percent acid activated bleaching clay for 20 minutes at 110° Celsius increases the enzyme's half life. Ten Brink et al. in U.S. Pat. Application No. 2005/0019316 further report, however, that such prior attempts to prolong the half life of a catalyst by purification of the lipids have been realized only on small scale laboratory processes, and that such processes have always failed when upgraded to an industrial scale. To address this concern, Ten Brink et al. disclose a method of treating “bleached” glyceride fats with a “bleaching earth zeolite” under high shear energy of 0.5 to 2.5 W/kg for a duration ranging from 5 minutes to 12 hours at a temperature range of 30 to 150° Celsius before exposing the lipid to a lipase catalyst for interesterification.
Other enzymatic treatments of lipid compositions are known. In addition to a lipase, enzymes of interest can include esterase; acylase; those enzymes that facilitate acidolysis reactions, transesterification reactions, ester synthesis, or ester interchange reactions; enzymes having phospholipase or protease activity, including thermostable and thermotolerant hydrolase activity; and polynucleotides.
It is thus an object of the invention to provide a process and apparatus for the continuous enzymatic treatment of a lipid-containing composition in multiple reaction modules connected in series, wherein the process can proceed continuously even if one of the modules has to be taken off-line for replacement of replenishment of the treatment medium.
It is thus another object of the invention to provide a process and apparatus for the continuous enzymatic treatment of a lipid-containing composition, in which the activity of the enzymes is prolonged.
It is another object of the invention to provide a process and apparatus for the continuous enzymatic treatment of a lipid-containing composition in multiple fixed bed reactors connected in series, wherein a fixed bed reactor can be replaced or replenished while the process remains at a substantially constant flow rate.
It is yet another object of the invention to provide a process and apparatus for continuous enzymatic treatment of a lipid-containing composition in multiple fixed bed reactors connected in series, wherein substantially all of the activity of a quantity of enzyme can be utilized before that quantity of enzyme is replaced or replenished.
It is yet another object of the invention to provide a process and apparatus for the enzymatic treatment of a lipid-containing composition in which the composition does not have to be deodorized prior to enzymatic treatment.
It is yet another object of the invention to provide a process and apparatus for the enzymatic treatment of a lipid-containing composition which requires only limited monitoring of the treatment process.
It is yet another object of the invention to provide a process and apparatus for the enzymatic treatment of a lipid-containing composition that is capable of producing a lipid-containing product meeting predetermined product specifications.
It is yet another object of the invention to provide a process and apparatus for the enzymatic treatment of a lipid-containing composition in multiple fixed bed reactors connected in series in which the flow rate remains substantially constant and is capable of producing a lipid-containing product meeting predetermined product specifications.